Nitric oxide is an endogenous vasodilator with an important role in the control of vascular smooth muscle cell tone. Nitric oxide and related nitrovasodilators act by stimulating guanylate cyclase and elevating cyclic GMP (cGMP), but the mechanism by which increased cGMP leads to vasodilation is not fully understood. To identify potential cGMP- binding proteins in VSMC, I have constructed and screened a human coronary artery smooth muscle cell cDNA library and isolated a novel 3.7 Kb cDNA clone that encodes a novel cDNA for a putative cGMP-gated ion channel. This proposal seeks to test the hypothesis that this cDNA encodes a functional ion channel important to the physiology of cGMP- mediated VSMC relaxation. Aim I. Functional expression and characterization of the Human VSMC cGMP-gated channel cDNA : The electrophysiological properties of the cloned channel will be studied by patch-clamp recordings of transfected cells to define the ligand specificity, ion selectivity, and pharmacologic response to known channel blockers. In addition, mutant channels with altered cGMP- binding domain and pore region will be generated by site-directed mutagenesis and antibodies to the CASMC channel will be prepared for use in subsequent functional studies. Aim II. Identification and characterization of the native cGMP-gated channel in VSMC: Northern blot analysis and in situ hybridization will be used to determine both the pattern of channel expression in tissues and its cellular localization. Patch-clamp recordings will then be used to characterize the native channel in freshly isolated vascular smooth muscle cells and to understand any functional differences between the recombinantly expressed channel and the channel in its native milieu. Aim III. Physiological role of the cGMP-gated channel in VSMC function: The physiological role of the cGMP-gated channel in vascular tone will be explored by (i) electrophysiologic studies of vascular smooth muscle cells in which the cloned CNG channel or mutants of the channel are overexpressed; and (ii) studies using an ex vivo vascular ring model and channel-specific inhibitors, as well as antibodies and channel- derived peptides. Finally, wild-type and dominant-negative mutant forms of the channel will be introduced into vessels of intact animals by adenoviral methods and the relaxation properties of blood vessel then will be studied ex vivo with the vascular ring model. These studies will explore a new pathway for cGMP regulation of vascular tone and are thus directly relevant to the study of vascular diseases and their treatment.